This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ***Please note the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** Work in year 4 of the COBRE was focused on exploring new avenues for human cryptosporidiosis research as well as development of new immunology assays to compliment this upcoming work. Establishment of functional immunology assays: Using specimens from clinical research studies on Cryptosporidium and live attenuated S. Typhi vaccines, we have established functional antibody and cellular assays for evaluation of human pathogens and toward standardization for use in a future human immunology core facility. Four new functional antibody assays have been developed using S. typhi as a model pathogen: opsonization/ phagocytosis (Figure 1);bacterial killing post-phagocytosis;measures of phagocytic index (PI, Figure 2);and a lysosome-colocalization assay. For cellular immunology, we have developed a seven-color flow cytometry assay to evaluate intracellular cytokines pre-and post-infection or vaccination (Figure 3). A B-cell memory ELISPOT assay has also been formally developed. All these assays are now in use for studying live attenuated dengue vaccines and will be used for future study of Cryptosporidium infection. Other assays, specifically for the study of viral pathogens, have been developed. These include standardized plaque reduction and neutralization assays (PRNT) using Vero cells (mosquito cell line) for Dengue serotype 1-4, West Nile, St. Louis Encephalitis and Yellow Fever viruses. Viral amplication assays (Dengue serotypes 1-4) have also been developed. New Cryptosporidium studies: opportunities for further Cryptosporidium work are anticipated to come to fruition in year 5 of the COBRE. First, opportunities for Cryptosporidium immunology are available in Dhaka, Bangladesh as part of a large birth cohort of children. Presently, 400 children have been enrolled and 12% have evidence of Cryptosporidium infection by 6 months of life. Clinical data, human DNA, peripheral lymphocytes and sera are available, making this an ideal setting for continuing our work into human immune responses to this infection. The work from this cohort will focus on three major avenues: genome-wide associations to expand our observations of genetic susceptibility to cryptosporidiosis;expanded and functional work on mannose-binding lectin (MBL) and other collectins/surfactants which function as an innate components in immunity to Cryptosporidium infection and evaluation of cellular immune responses to infection using 7 or 8-color flow cytometry and ELISPOT assays (see above).Secondly, we are initiating enrollment of a large cohort of persons with end-stage AIDS and Cryptosporidium infection (Uganda). We will be confirming the MBL association with Cryptosporidium in this population.